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A kind of method for low pH virus inactivation on the column

A virus inactivation, column volume technology, applied in the biological field, can solve the problems of incomplete virus inactivation, long adjustment process time, hanging on the wall, etc., to achieve the effect of stabilizing protein products, improving process robustness, and high adsorption capacity

Active Publication Date: 2022-04-05
HUZHOU TEACHERS COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Low pH virus inactivation usually needs to be carried out at a lower pH, and local overacids are prone to occur during acid adjustment, resulting in increased aggregate content in some products
Secondly, in large-scale production, the pH adjustment operation is relatively cumbersome, the adjustment process takes a long time, and the consistency of pH adjustment is not easy to control
In addition, low pH adjustment in the container is prone to dead corners or residual liquid hanging on the wall, which may lead to incomplete virus inactivation

Method used

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  • A kind of method for low pH virus inactivation on the column

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Take a Capto S ImpAct cation chromatography column with a column volume of 10 mL, and wash the cation chromatography column with 50 mM citric acid equilibration buffer at pH 4.0 for 3 column volumes. Take a feed solution containing 0.2 g of monoclonal antibody, adjust the pH to 4.0 with citric acid or sodium citrate, and obtain a protein loading solution. The protein sample solution was loaded onto the cation chromatography column with a sample volume of 20 g / L filler. Flush the cation chromatography column for 3 column volumes with 50 mM citric acid equilibration buffer, pH 4.0. Flush the cation chromatography column for 3 column volumes with 50 mM citric acid inactivation buffer, pH 3.0, and pause for 30 min. The recovered product was eluted with 50 mM citric acid elution buffer containing 100 mM NaCl at pH 5.0, and the eluted fractions were collected to obtain the monoclonal antibody product after low pH virus inactivation. The purity of electrophoresis analysis was...

Embodiment 2

[0024] Take a Capto S ImpAct cation chromatography column with a column volume of 35 mL, and wash the cation chromatography column with 50 mM citric acid equilibration buffer at pH 6.0 for 8 column volumes. The feed solution containing 4.2 g of bispecific antibody was taken, and the pH was adjusted to 6.0 with citric acid or sodium citrate to obtain a protein loading solution. The protein sample solution was loaded onto the cation chromatography column with a sample volume of 120 g / L filler. Flush the cation chromatography column for 8 column volumes with 50 mM citric acid equilibration buffer, pH 6.0. Flush the cation chromatography column for 8 column volumes with 50 mM citric acid inactivation buffer, pH 3.8, and pause for 120 min. The recovered product was eluted with 50 mM citric acid elution buffer containing 300 mM NaCl at pH 6.0, and the eluted fractions were collected to obtain a low-pH virus-inactivated bispecific antibody product with a purity of 98.5% by electroph...

Embodiment 3

[0026] Take a Poros XS cation chromatography column with a column volume of 20 mL, and wash the cation chromatography column with 50 mM citric acid equilibration buffer at pH 5.0 for 4 column volumes. Take a feed solution containing 2.0 g of monoclonal antibody, adjust the pH to 5.0 with citric acid or sodium citrate, and obtain a protein loading solution. The protein sample solution was loaded onto the cation chromatography column, and the sample volume was 100 g / L filler. Flush the cation chromatography column for 8 column volumes with 50 mM citric acid equilibration buffer, pH 5.0. Flush the cation chromatography column for 4 column volumes with 50 mM citric acid inactivation buffer, pH 3.5, and pause for 90 min. The recovered product was eluted with 50 mM citric acid elution buffer at pH 5.0 containing 200 mM NaCl, and the eluted fractions were collected to obtain the monoclonal antibody product after low pH virus inactivation. The purity of electrophoresis analysis was 9...

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Abstract

The invention belongs to the field of biotechnology, and specifically discloses a method for virus inactivation at low pH on a column. The specific steps are as follows: 1) Equilibrate the cation chromatography column. 2) Protein sample loading. 3) Wash the cation chromatography column after loading. 4) Low pH virus inactivation on the column. 5) Wash the cation chromatography column again. 6) Protein elution and recovery. The feature of the present invention is to develop a fast and convenient low-pH virus inactivation method. The key of the method is to directly carry out low-pH virus inactivation on the cation chromatography column. The operation steps are simple, the process is robust, and it is beneficial to protect the acid-sensitive protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a low-pH virus inactivation method on a column in the downstream production process of protein drugs. Background technique [0002] In recent years, protein drugs have attracted more and more attention. With the development of mammalian cell culture technology, a large number of antibody proteins or fusion proteins are expressed and produced in mammals. At present, Chinese hamster ovary cells (CHO, Chinese hamster ovary cells) are one of the most widely used expression systems. Products produced by expression in animal cells have the risk of introducing viruses. In order to ensure the safety of the final product, the downstream production process is required to effectively remove or inactivate potential viruses. [0003] Low pH virus inactivation is a common virus inactivation method, which can effectively reduce the virus level, but there are still some shortcomings i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/18
CPCA61L2/0011C07K1/18A61L2202/21
Inventor 顾佳黎
Owner HUZHOU TEACHERS COLLEGE